Device for the sustained release of aggregation-stabilized, biologically active agent

ABSTRACT

A device for the sustained release in vivo of a water soluble, biologically active agent wherein the agent is susceptible to aggregation comprising a drug delivery device and aggregation-stabilized, biologically active agent wherein the aggregation-stabilized agent is disposed within the drug delivery device.

RELATED APPLICATIONS

[0001] This application is a Continuation of co-pending U.S. patentapplication Ser. No. 08/934,830, filed Sep. 22, 1997, which is aContinuation of U.S. patent application Ser. No. 08/521,744, filed Aug.31, 1995, now abandoned, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 08/765,558, which is the U.S. National Stage ofInternational Application No.: PCT/US95/07348 filed Jun. 7, 1995,published in English, which is a Continuation-in-Part of U.S.application Ser. No. 08/279,784 filed Jul. 25, 1994, now U.S. Pat. No.5,711,968. This application is also a Continuation-in-Part of U.S.patent application Ser. No. 08/478,502, filed Jun. 7, 1995, now U.S.Pat. No. 5,716,644 which is a Continuation-in-Part of U.S. patentapplication Ser. No. 07/885,307, filed Jun. 11, 1992, now abandoned;U.S. patent application Ser. No. 08/483,318, filed Jun. 7, 1995, nowU.S. Pat. No. 5,674,534, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 07/885,307, filed Jun. 11, 1992, now abandoned;U.S. patent application Ser. No. 08/473,544 filed Jun. 7, 1995, now U.S.Pat. No. 5,654,010, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 07/984,323, filed Dec. 2, 1992, now abandoned; andU.S. patent application Ser. No. 08/477,725, filed Jun. 7, 1995, nowU.S. Pat. No. 5,667,808, which is a Continuation-in-Part of U.S.application Ser. No. 07/984,323, filed Dec. 2, 1992, now abandoned. Theentire teachings of the above application(s) are incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] Many illnesses or conditions require administration of a constantor sustained level of a medicament or biologically active agent toprovide the most effective prophylactic or therapeutic. This may beaccomplished through a multiple dosing regimen or by employing a systemthat releases the medicament in a sustained fashion.

[0003] Attempts to sustain medication levels include the use ofbiodegradable materials, such as polymeric matrices, containing themedicament. The use of these matrices, for example, in the form ofmicroparticles or microcarriers, provides an improvement in thesustained release of medicaments by utilizing the inherentbiodegradability of the polymer to control the release of the medicamentand provide a more consistent, sustained level of medication andimproved patient compliance.

[0004] However, these sustained release devices often exhibited highinitial bursts of agent release and minimal agent release thereafter.Further, due to the high solution concentration of agent within andlocalized around these sustained release devices, the agent moleculeshave tended to aggregate thereby increasing immunogenicity in vivo andinterfering with the desired release profile for the agent.

[0005] Therefore, a need exists for a means for sustaining the releaseof a biologically active agent in vivo without significant aggregateformation and thus with a reduced immune response to the agent over therelease period of the agent.

SUMMARY OF THE INVENTION

[0006] This invention relates to a device for the sustained release invivo of a water soluble, biologically active agent wherein said agent issusceptible to aggregation, comprising a drug delivery device andaggregation-stabilized, biologically active agent wherein theaggregation-stabilized agent is disposed within the drug deliverydevice.

[0007] There are many advantages to this sustained release device for abiologically active agent. These advantages include longer, moreconsistent in vivo blood levels of the agent, lower initial bursts ofthe agent, and increased therapeutic benefits by eliminatingfluctuations in serum agent levels. The advantages also include betterretention of biological activity of the agent and reduced immunogenicitywhen in vivo. The advantages further include more complete release of anagent from a sustained release device.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

[0009]FIG. 1 is a plot of a) the cumulative release of monomericerythropoietin (EPO), b) the cumulative release of EPO (monomer EPO plusaggregated EPO), and c) the percentage of EPO which is released as amonomer during the interval between an indicated time point and theimmediately preceding time point, in vitro in HEPES buffer, frommicrocarriers of unblocked poly(lactide-co-glycolide) polymer (PLGA)(10,000 Dalton MW), containing 10% (w/w) MgCO₃ and 5% (w/w) of the Am1formulation of Example 6, versus time over a 28 day interval.

[0010]FIG. 2 is a plot of a) the cumulative release of monomeric EPO, b)the cumulative release of EPO (monomer plus aggregate), and c) thepercentage of EPO which is released as a monomer during the intervalbetween an indicated time point and the immediately preceding timepoint, in vitro in HEPES buffer, from microcarriers unblocked PLGA(10,000 Dalton MW), containing 10% (w/w) MgCO₃ and 5% (w/w) of the Am7formulation of Example 6, versus time over a 28 day interval.

[0011]FIG. 3 is a plot of a) the cumulative release of monomeric EPO, b)the cumulative release of EPO (monomer plus aggregate), and c) thepercentage of EPO which is released as a monomer during the intervalbetween an indicated time point and the immediately preceding timepoint, in vitro in HEPES buffer, from microcarriers of blocked PLGA(10,000 Dalton MW), containing 10% (w/w) ZnCO₃ and 10% (w/w) of the Zn1formulation of Example 6, versus time over a 28 day interval.

[0012]FIG. 4 is a plot of the serum concentration (IU/ml) ofInterferon-α,2b (IFN-α,2b) in rats, which were subcutaneouslyadministered IFN-α,2b controlled release formulated microcarriers ofExample 2, versus time over a 6 day interval.

[0013]FIG. 5 is a plot of the serum concentration (IU/ml) of IFN-α,2b inrats, which were subcutaneously administered IFN-α,2b controlled releaseFormula 2 microcarriers of Example 2, versus time over a 6 day interval.

[0014]FIG. 6 is a plot of the serum concentration (IU/ml) of IFN-α,2b inrats, which were subcutaneously administered IFN-α,2b controlled releaseFormula 3 microcarriers of Example 2, versus time over a 7 day interval.

[0015]FIG. 7 is a plot of the serum concentration (IU/ml) of IFN-α,2b inrats, which were subcutaneously administered IFN-α,2b controlled releaseFormula 4 microcarriers of Example 2, versus time over a 7 day interval.

[0016]FIG. 8 is a plot of the serum concentration (IU/ml) of IFN-α,2b,in rats, which were subcutaneously administered IFN-α,2b controlledrelease Formula 5 microcarriers of Example 2, versus time over a 7 dayinterval.

[0017]FIG. 9 is a plot of the serum concentration (IU/ml) of IFN-α,2b inrats, which were subcutaneously administered IFN-α,2b controlled releaseFormula 6 microcarriers of Example 2, versus time over a 7 day interval.

[0018]FIG. 10 is a plot of the serum concentration (IU/ml) of IFN-α,2bversus time over a 7 day interval in rats which were subcutaneouslyadministered IFN-α,2b controlled release Formula 7 microcarriers ofExample 2 having a 1:1 zinc carbonate-to-IFN-α,2b ratio.

[0019]FIG. 11 is a plot of the serum concentration (IU/ml) of IFN-α,2bversus time over a 29 day interval in rats which were subcutaneouslyadministered a) IFN-α,2b controlled release microcarriers of Formula 8of Example 2, wherein the rats were immunosuppressed with cyclosporin Aand hydrocortisone (two groups) and b) the same formulation of IFN-α,2bcontrolled release microcarriers wherein the rats were notimmunosuppressed.

[0020]FIG. 12 is a plot of the serum concentrations (IU/ml) of IFN-α,2bversus time over a 14 day interval in monkeys which were subcutaneouslyadministered a) IFN-α,2b controlled release microcarriers of Example 2having a 1:8 zinc carbonate to IFN-α,2b ratio and b) an equal dose ofIFN-α,2b in 0.9% saline solution.

[0021]FIG. 13 is a plot of the serum concentration (ng/ml) of hGH versustime over a 28 day interval in rats which were subcutaneouslyadministered a) aggregation-stabilized hGH microcarriers of 31Kunblocked PLGA containing 1% ZnCO₃ of Example 5 wherein the rats wereimmunosuppressed with cyclosporin A and hydrocortisone and b) the samehGH microcarriers wherein the rats were not immunosuppressed.

[0022]FIG. 14 is a plot of the serum concentration (ng/ml) of hGH versustime over a 28 day interval in rats which were subcutaneouslyadministered a) aggregation-stabilized hGH microcarriers of 8K unblockedPLGA containing 1% ZnCO₃ of Example 5 wherein the rats wereimmunosuppressed with cyclosporin A and hydrocortisone and b) the samehGH microcarriers wherein the rats were not immunosuppressed.

[0023]FIG. 15 is a plot of the serum concentration (ng/ml) of hGH versustime for a 61 day interval in monkeys which were subcutaneouslyadministered aggregation-stabilized hGH microcarriers of Example 5containing 15% hGH (complexed with Zn⁺² at a 6:1 Zn⁺²:hGH molar ratio),6% w/w ZnCO₃ and 10K blocked PLGA.

[0024]FIG. 16 is a plot of the serum concentration (ng/ml) of hGH versustime for a 60 day interval in monkeys which were subcutaneouslyadministered aggregation-stabilized hGH microcarriers of Example 5containing 15% hGH (complexed with Zn⁺² at a 6:1 Zn⁺²:hGH molar ratio),1% w/w ZnCO₃ and 8K unblocked PLGA.

[0025]FIG. 17 is a plot of the serum concentration (ng/ml) of hGH versustime for a 68 day interval in monkeys which were subcutaneouslyadministered aggregation-stabilized hGH microcarriers of Example 5containing 15% hGH (complexed with Zn⁺² at a 6:1 Zn⁺²:hGH molar ratio),1% w/w ZnCO₃ and 31K unblocked PLGA.

[0026]FIG. 18 is a plot of the serum concentration (ng/ml) of hGH andIGF-1 versus time for a 32 day interval in monkeys which weresubcutaneously administered aggregation-stabilized hGH microcarriers ofExample 16 in 8K unblocked PLGA.

[0027]FIG. 19 is a plot of the serum concentration (ng/ml) of hGH versustime for 30 and 39 day intervals for a) aggregation-stabilized hGH 8Kunblocked PLGA microcarriers and b) daily aqueous hGH injections,respectively.

[0028]FIG. 20 is a plot of the percent reticulocytes in blood ofcyclosporin/hydrocortisone (CS/HC) treated and untreated rats, whichwere subcutaneously injected with 10,000 units of the EPO sustainedrelease microcarriers RMAm7, described in Example 17 a bolus of 2,000units of aqueous EPO, administered on day 28, respectively, versus timeover a 36 day interval.

[0029]FIG. 21 is a plot of the serum concentration (IU/ml) of EPO inrats, which were subcutaneously administered various EPO sustainedrelease microcarriers, described in Example 6, versus time over a 22 dayinterval.

[0030]FIG. 22 is a plot of the percent reticulocytes in blood of rats,which were subcutaneously injected with 10,000 units of various EPOsustained release microcarriers, described in Example 6, versus timeover a 28 day interval.

[0031]FIG. 23 is a plot of the serum concentration (IU/ml) of IFN-α,2bversus time over a 7 day interval in rats which were subcutaneouslyadministered three different IFN-α,2b controlled release microcarriersof Example 2 having zinc carbonate to IFN-α,2b ratios of 1:1, 3:1 and8:1.

DETAILED DESCRIPTION OF THE INVENTION

[0032] A biologically active agent, as defined herein, is an agent, orits pharmaceutically acceptable salt, which is in its molecular,biologically active form when released in vivo, thereby possessing thedesired therapeutic and/or prophylactic properties in vivo. Biologicallyactive agents suitable for the composition and method of the inventionare agents which are soluble in aqueous solutions and biological fluidsand which are susceptible to aggregation in vivo. Examples of suitablebiologically active agents include proteins such as immunoglobulin-likeproteins, antibodies, cytokines (e.g., lymphokines, monokines,chemokines), interleukins, interferons, erythropoietin, nucleases, tumornecrosis factor, colony stimulating factors, insulin, enzymes, tumorsuppressors, hormones (e.g., growth hormone and adrenocorticotropichormone), antigens (e.g., bacterial and viral antigens) and growthfactors; peptides such as protein inhibitors; nucleic acids, such asantisense molecules; oligonucleotides; and ribozymes.

[0033] A sustained release of a biologically active agent is a releasewhich results in biologically effective serum levels of the biologicallyactive, molecular (monomeric or non-aggregated) form of the agent over aperiod longer than that obtained following direct administration of anaqueous solution of the agent. A biologically effective serum level ofan agent is a level which will result in the desired biological responsewithin the recipient. Usually, in a sustained release, the serum levelof the agent is above endogenous levels. Typically, a sustained releaseof an agent is for a period of a week or more, and preferably for twoweeks or more.

[0034] A sustained release of non-aggregated, biologically active agentcan be a continuous or non-continuous release with relatively constantor varying rates of release from a drug delivery device. The continuityof release of the biologically active agent can be affected by theloading of the agent, selection of excipients to produce the desiredeffect, and/or by other conditions such as the type of polymer used ifthe biologically active agent is encapsulated within a polymeric matrix.

[0035] A drug delivery device, as defined herein, includes anycomposition, such as diffusion-controlled polymeric and protein systemsof the reservoir or matrix-type, or systems such as pressure-drivenosmotic or syringe pumps wherein the rate of release of a biologicallyactive agent is sustained by use of a drug delivery device to releasesaid agent in vivo.

[0036] Aggregation-stabilized biologically active agent, as definedherein comprises a suitable agent in its biologically active, molecular(monomeric) form wherein the biologically active agent is stabilizedagainst aggregation during formation of the sustained release device andwhile the device is employed in vivo. A biologically active agent can beaggregation-stabilized by several means, such as by controlling thesolubilization of the agent in vivo and by controlling the environmentalconditions experienced by the agent during device formation and in vivo.These means are typically dependent upon the specific biologicallyactive agent to be aggregation-stabilized. Preferably, the means foraggregation-stabilizing a biologically active agent should not convertthe agent to a form that will reduce in vivo biological activity such asby oxidation.

[0037] An aggregation-stabilized biologically active agent is stabilizedagainst significant aggregation in vivo over the sustained releaseperiod. Significant aggregation is defined as an amount of aggregationthat will reduce or preclude the achievement of effective serum levelsin vivo of the biologically active agent over the sustained releaseperiod. Typically, significant aggregation is aggregation of about 10%or more of the original amount of biologically active agent in thesustained drug delivery device. Preferably, aggregation is maintainedbelow about 5% of the initial loading of the molecular form of theagent. More preferably, aggregation is maintained below about 2% of theinitial loading of biologically active agent.

[0038] In one embodiment of the sustained release device of the presentinvention, the biologically active agent is mixed with anaggregation-stabilizer wherein the in vivo solubilization of thebiologically active agent is controlled. Typically anaggregation-stabilizer reduces the solubility of the biologically activeagent, precipitates out a salt of the agent or forms a complex of theagent. The aggregation-stabilizer and the biologically active agent canbe separately contained within the sustained drug delivery device, suchas a device containing particles of aggregation-stabilizer and separateparticles of biologically active agent, and/or can be combined togetherin complexes or particles which contain both the aggregation-stabilizerand the biologically active agent.

[0039] The suitability of candidate aggregation-stabilizers forstabilizing a biologically active agent against aggregation can bedetermined by one of ordinary skill in the art by performing a varietyof stability indicating techniques such as SEC, polyacrylamide gelelectrophoresis (PAGE) and potency tests on protein obtained fromparticles containing the aggregation-stabilized agent and for theduration of release from the sustained release device, as described inExample 5 for hGH and Examples 8-9 for EPO.

[0040] Suitable particles of aggregation-stabilized biologically activeagent are solid particles, including lyophilized particles, freeze-driedparticles, pressed pellets, and particles formed by any other meansknown in the art for forming a solid particle from a mixture of twocomponents (e.g., biologically active agent and an aggregationstabilizer) wherein one component is temperature sensitive.

[0041] The amount of an agent which is contained in a sustained releasedevice containing biologically active, aggregation-stabilized particlesof the agent is a therapeutically or prophylactically effective amountwhich can be determined by a person of ordinary skill in the art takinginto consideration factors such as body weight, condition to be treated,type of device used, and release rate from the device.

[0042] In one example of this embodiment wherein the in vivosolubilization of a biologically active agent is controlled, abiologically active agent is aggregation-stabilized when mixed with atleast one type of metal cation from a metal cation component, which isthe aggregation-stabilizer, wherein the agent is complexed and/orcomplexes in vivo with the metal cation to aggregation-stabilize theagent.

[0043] Suitable aggregation-stabilizing metal cations includebiocompatible metal cations which will not significantly oxidize theagent. Typically, oxidation of a biologically active agent by a metalcation is not significant if this oxidation results in a loss of theagent's potency of about 10% or less. A metal cation component isbiocompatible if it is non-toxic to the recipient in the quantitiesused, and also presents no significant deleterious or untoward effectson the recipient's body, such as an immunological reaction at theinjection site. Preferably, the metal cation is multivalent.

[0044] Examples of suitable aggregation-stabilizing metal cationsinclude cations of non-transition metals, such as Mg⁺² and Ca⁺².Suitable aggregation-stabilizing metal cations also include cations oftransition metals, such as Cu⁺², Co⁺², Fe⁺³ and Ni⁺². In a preferredembodiment, Zn⁺² is used as an aggregation-stabilizing metal cation. Thesuitability of metal cations for stabilizing a biologically active agentcan be determined by one of ordinary skill in the art by performing avariety of stability indicating techniques such as polyacrylamide gelelectrophoresis, isoelectric focusing, reverse phase chromatography,size exclusion chromatography (SEC) and potency tests on particles ofthe biologically active agent containing metal cations to determine thepotency of the agent after particle formation, such as bylyophilization, and for the duration of release from microparticles.

[0045] It is preferred that the metal cation and biologically activeagent are complexed within the sustained drug delivery device beforeadministration to a subject.

[0046] It is also preferred that the mixture of the metal cation and thebiologically active agent are in the form of solid particles, morepreferably, lyophilized particles.

[0047] The molar ratio of metal cation to biologically active agent istypically between about 1:2 and about 100:1, and is preferentiallybetween about 2:1 and about 10:1.

[0048] The use of metal cations to form aggregation-stabilized particlesof the biologically active agents, interferon (IFN) and human growthhormone (hGH), are further described in Examples 1 and 4. In addition,the formation of sustained release devices of polymeric microcarrierscontaining metal cation-stabilized IFN or hGH are described in Examples2 and 5. Furthermore, the aggregation-stabilization efficacy of metalcations complexed with IFN or hGH, within lyophilized particlesdispersed in polymeric microcarriers, over a sustained release period invivo are described in Examples 10-12 or Examples 13-16, respectively.

[0049] The use of additional metal cations, dispersed within thepolymeric matrix of a sustained release device, to furtheraggregation-stabilize a biologically active agent (hGH or IFN) aredescribed in Examples 14 and 18.

[0050] The polymeric matrix is believed to function as a reservoir ofmetal cations so that the formation of cation-complexed protein isfavored and dissociation into soluble protein is disfavored. Wherein theaqueous solubility of the metal cation component in the polymeric matrixis low, the release of metal cations from the matrix is slow, thusmodulating the solubility of the protein.

[0051] In another example of the embodiment wherein the solubility of abiologically active agent is reduced by an aggregation stabilizer, thebiologically active agent is mixed with an aggregation stabilizer whichreduces solubility by precipitating the agent from the aqueous solution,thereby maintaining a suitably low localized concentration of solubleagent below a concentration at which significant aggregation occurs. Alocalized concentration of an agent is the concentration of solvatedagent within, between or immediately surrounding the sustained releasedevice. Suitable materials for precipitating an agent, such as aprotein, without denaturing the agent, include salts which are in theHofmeister series of precipitants of serum globulins (or “salting-outsalts”) as described by Thomas E. Creighton in Proteins: Structures andMolecular Principles, p149-150 (published by W.H. Freeman and Company,New York). Suitable salting-out salts for use in this invention include,for example, salts containing one or more of the cations Mg⁺², Li⁺, Na⁺,K⁺ and NH₄ ⁺; and also contain one or more of the anions S₄ ⁻², HPO₄ ⁻²,acetate, citrate, tartrate, Cl⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻ and SCN⁻.

[0052] Again, the biologically active agent and the precipitant can becombined within particles and/or can be separately contained within thesustained release device. Preferably, a biologically active agent and aprecipitant are combined in a lyophilized particle. The formation oflyophilized particles containing the agent erythropoietin and aprecipitant, and the use of these particles in polymeric microcarriersustained release devices, are described in Examples 6 and 7. Theefficacy of precipitants in preventing aggregation of EPO in vitro andin vivo over a sustained period are also described in Examples 8-9 andExample 17, respectively.

[0053] In yet another embodiment for stabilizing a biologically activeagent against aggregation, the agent is mixed with a buffer which willmaintain the agent under pH conditions in vivo that can affect the rateof solubilization of the agent and/or prevent the formation in vivo ofbiologically inactive or insoluble forms (precipitates or gels which areinsoluble in vivo) of the agent. Examples of such buffers include, forinstance, phosphate buffers.

[0054] A preferred sustained release device of the present invention isa biocompatible polymeric matrix containing particles of anaggregation-stabilized biologically active agent dispersed therein.Polymers suitable to form a polymeric matrix of a sustained releasedevice of this invention are biocompatible polymers which can be eitherbiodegradable or non-biodegradable polymers, or blends or copolymersthereof.

[0055] A polymer, or polymeric matrix, is biocompatible if the polymer,and any degradation products of the polymer, are non-toxic to therecipient and also present no significant deleterious or untowardeffects on the recipient's body, such as an immunological reaction atthe injection site.

[0056] Biodegradable, as defined herein, means the composition willdegrade or erode in vivo to form smaller chemical species. Degradationcan result, for example, by enzymatic, chemical and/or physicalprocesses. Suitable biocompatible, biodegradable polymers include, forexample, poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polyacetals,polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,biodegradable polyurethanes, blends and copolymers thereof.

[0057] Biocompatible, non-biodegradable polymers suitable for asustained release device include non-biodegradable polymers selectedfrom the group consisting of polyacrylates, polymers of ethylene-vinylacetates and other acyl substituted cellulose acetates, non-degradablepolyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinyl imidazole), chlorosulphonate polyolefins, polyethylene oxide,blends and copolymers thereof.

[0058] Further, the terminal functionalities of a polymer can bemodified. For example, polyesters can be blocked, unblocked or a blendof blocked and unblocked polyesters. A blocked polyester is asclassically defined in the art, specifically having blocked carboxyl endgroups. Generally, the blocking group is derived from the initiator ofthe polymerization and is typically an alkyl group. An unblockedpolyester is as classically defined in the art, specifically having freecarboxyl end groups.

[0059] Acceptable molecular weights for polymers used in a sustainedrelease device can be determined by a person of ordinary skill in theart taking into consideration factors such as the desired polymerdegradation rate, physical properties such as mechanical strength, andrate of dissolution of polymer in solvent. Typically, an acceptablerange of molecular weights is of about 2,000 Daltons to about 2,000,000Daltons. In a preferred embodiment, the polymer is a biodegradablepolymer or copolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide) (hereinafter “PLGA”) with a lactide:glycolideratio of about 1:1 and a molecular weight of about 5,000 Daltons toabout 70,000 Daltons. In an even more preferred embodiment, themolecular weight of the PLGA used in the present invention has amolecular weight of about 5,000 Daltons to about 42,000 Daltons.

[0060] Typically, a polymeric sustained release microcarrier willcontain from about 0.01% (w/w) to approximately 50% (w/w) ofaggregation-stabilized biologically active agent (dry weight of thecomposition). The amount of agent used will vary depending upon thedesired effect of the agent, the planned release levels, and the timespan over which the agent will be released. A preferred range of agentloading is between about 0.1% (w/w) to about 30% (w/w) agent. A morepreferred range of agent loading is between about 0.5% (w/w) to about20% (w/w) agent.

[0061] In another embodiment, a polymeric sustained release compositionalso contains a biocompatible metal cation component, which is notcontained in the biologically active, aggregation-stabilized particles,but is dispersed within the polymer. The metal cation of this metalcation component acts to modulate the release of the biologically activeagent from the polymeric sustained release composition.

[0062] This metal cation component typically comprises at least one typeof multivalent metal cations. A metal cation component, as definedherein, is a component containing at least one kind of multivalent metalcation (having a valency of +2 or more) in a non-dissociated state, adissociated state, or a combination of non-dissociated and dissociatedstates. Suitable metal cation components include, for instance, metalsalts, metal hydroxides, and basic (pH of about 7 or higher) salts ofweak acids wherein the salt contains a metal cation. It is preferredthat the metal cation be divalent. Examples of metal cation componentssuitable to modulate release of a biologically active agent, include, orcontain, for instance, Mg(OH)₂, MgCO₃ (such as 4MgCO₃.Mg(OH)₂.5H₂O),ZnCO₃ (such as 3Zn(OH)₂.2ZnCO₃), CaCO₃, Zn₃(C₆H₅O₇)₂, Mg(OAc)₂, MgSO₄,Zn(OAc)₂, ZnSO₄, ZnCl₂, MgCl₂ and Mg₃(C₆H₅O₇)₂. A suitable ratio ofmetal cation component-to-polymer is between about 1:99 to about 1:2 byweight. The optimum ratio depends upon the polymer and the metal cationcomponent utilized.

[0063] The metal cation component can optionally contain cation speciesand/or anion species which are contained in an aggregation stabilizerwithin particles of the agent. The metal cation component acts tomodulate the release of the agent from the polymeric matrix of thesustained release composition and can also enhance the stability ofagent in the composition against aggregation. In a modulated release, atleast one release characteristic of the agent, such as the initialrelease level, the subsequent release levels, duration of release and/orthe amount of agent released, is different from the releasecharacteristics exhibited by the agent being released from a polymericmatrix, wherein the polymeric matrix does not contain a dispersed metalcation component.

[0064] A polymeric matrix containing a dispersed metal cation componentto modulate the release of a biologically active agent from thepolymeric matrix is further described in co-pending U.S. patentapplication Ser. No. 08/237,057, filed May 3, 1994 and co-pending PCTPatent Application PCT/US95/05511, the teachings of which areincorporated herein by reference in their entirety.

[0065] In yet another embodiment, at least one pore forming agent, suchas a water soluble salt, sugar or amino acid, is included in a polymericmicroparticle to modify the microstructure of the microparticle. Theproportion of pore forming agent added to a polymer solution, from whichthe microparticle is formed, is between about 1% (w/w) to about 30%(w/w). It is preferred that at least one pore forming agent be includedin a nonbiodegradable polymeric matrix.

[0066] The biologically active agent in a sustained release device ofthe present invention can also contain other excipients, such asstabilizers and bulking agents. Stabilizers are added to maintain thepotency of the biologically active agent over the duration of theagent's release. Suitable stabilizers include, for example,carbohydrates, amino acids, fatty acids and surfactants and are known tothose skilled in the art. For amino acids, fatty acids andcarbohydrates, such as sucrose, lactose, mannitol, inulin, maltose,dextran and heparin, the mass ratio of carbohydrate to biologicallyactive agent is typically between about 1:10 and about 20:1. Forsurfactants, such as polysorbates (e.g., Tween™) and polyoxamers andpolyoxamines (e.g., Pluronic™), the mass ratio of surfactant to agent istypically between about 1:1000 and about 1:20.

[0067] Solubility agents can also be added to further modify thesolubility of the agent. Suitable solubility agents include complexingagents, such as albumin and protamine, which can be used to slow therelease rate of the agent from a polymeric matrix. The weight ratio ofsolubility agent to biologically active agent is generally between about1:99 and about 20:1.

[0068] Bulking agents typically comprise inert materials. Suitablebulking agents are known to those skilled in the art.

[0069] A polymeric sustained release composition of this invention canbe formed into many shapes such as a film, a pellet, a cylinder, a discor a microcarrier. A microcarrier, as defined herein, comprises apolymeric component having a diameter of less than about one millimeterand containing at least one particle of aggregation-stabilized,biologically active agent dispersed therein. A microcarrier can have aspherical, non-spherical or irregular shape. It is preferred that amicrocarrier be generally spherical in shape. Typically, themicrocarrier will be of a size suitable for injection. A preferred sizerange for microcarriers is from about 1 to about 180 microns indiameter, such as for injection through a 23-gauge needle.

[0070] In the method for preparing aggregation-stabilized agent, thebiologically active agent is mixed with a suitableaggregation-stabilizer. It is understood that either or both the agentand stabilizer can be in solid form, typically particulate, or dissolvedin an aqueous solution. It is preferred that the agent and stabilizer becombined in single particles, which are more preferably lyophilized.

[0071] In the embodiment wherein a biologically active agent is mixedwith a metal cation component to form particles, the agent is mixed in asuitable solvent with at least one suitable metal cation component toform a mixture, wherein each component of the mixture can be insuspension or solution, or a combination thereof. The concentration ofagent in solution is typically between about 0.1 to about 20 mg agent/mlof solvent, and preferentially, between about 1.0 to about 5.0 mgagent/ml of solvent.

[0072] In a preferred embodiment, the agent is contacted with at leastone suitable aggregation-stabilizing metal cation, such as Ca⁺² or Zn⁺²,and with a suitable solvent, under pH conditions suitable for forming acomplex of the metal cation and the agent. Typically, the complexedagent will be in the form of a cloudy precipitate, which is suspended inthe solvent. However, the complexed agent can also be in solution.

[0073] In an embodiment wherein particles of an agent stabilized with aprecipitant are formed, the agent is mixed in a suitable aqueous solventwith at least one suitable precipitant to form a stabilizing mixture,wherein each component of the stabilizing mixture can be in suspensionor solution, or a combination thereof.

[0074] In forming a stabilizing mixture, the content of precipitant istypically between about 10% (w/w) and about 80% (w/w) of the totalsolids in agent particles and is preferentially more than about 40%(w/w).

[0075] It is understood that the agent can be in a solid or a dissolvedstate, prior to being contacted with the aggregation stabilizer. It isalso understood that the aggregation stabilizer can be in a solid or adissolved state, prior to being contacted with the agent. In a preferredembodiment, a buffered aqueous solution of an agent is mixed with anaqueous solution of the aggregation stabilizer.

[0076] Suitable solvents are those in which the agent and the metalcation component are each at least slightly soluble, such as in anaqueous sodium bicarbonate buffer or in an aqueous phosphate buffer orcitrate buffer or combinations thereof. For aqueous solvents, it ispreferred that water used be either deionized water orwater-for-injection (WFI).

[0077] The stabilizing mixture is then dried, such as by lyophilization,to form particulate aggregation-stabilized agent. The stabilizingmixture can be bulk lyophilized or can be divided into smaller volumeswhich are then lyophilized. In a preferred embodiment, the stabilizingmixture is micronized, such as by use of an ultrasonic nozzle, and thenlyophilized to form aggregation-stabilized agent particles. Acceptablemeans to lyophilize the stabilizing mixture include those known in theart. A solid stabilizing mixture can be pressed into pellets.

[0078] A suitable pH range can be achieved by dialysis with a buffer, byusing the buffer as a solvent for the agent and/or aggregationstabilizer, and by making one or more bulk additions of buffer to theagent solution before, during, and/or after addition of the aggregationstabilizer.

[0079] The stabilizing mixture is usually buffered to a pH between about4.0 and about 8.0 to maintain pH in a range which will prevent asignificant loss of biological activity resulting from pH changes duringparticle formation and/or to support formation of complexes. A preferredpH range is between about 5.0 and about 7.4. Suitable pH conditions aretypically achieved through use of an aqueous buffer, such as sodiumbicarbonate, as the solvent for the agent and aggregation stabilizer.Typically, the content of buffer in a stabilizing mixture is betweenabout 0.1% (w/w) and about 20% (w/w) of total solids.

[0080] Preferably, particles of aggregation-stabilized agent are betweenabout 1 to about 6 micrometers in diameter. The agent particles can befragmented separately, as described in co-pending U.S. patentapplication Ser. No. 08/006,682, filed Jan. 21, 1993, which describes aprocess for producing small particles of biologically active agents,which is incorporated herein in its entirety by reference. Alternately,the agent particles can be fragmented after being added to a polymersolution, such as by means of an ultrasonic probe or ultrasonic nozzle.

[0081] The formation of Zn⁺²-stabilized IFN or hGH particles are furtherdescribed in Examples 1 and 4.

[0082] In one embodiment for forming a sustained release device, asuitable amount of aggregation-stabilized particles of agent is added toa polymer solution. The agent particles can be dispersed with thepolymer solution by stirring, agitation, sonication or by other knownmixing means. The polymer solution, having a dispersion of biologicallyactive, aggregation-stabilized agent is then solidified, by appropriatemeans, to form a sustained release composition of this invention.

[0083] Alternately, biologically active, aggregation-stabilizedparticles of agent and a polymer can be mixed into a polymer solventsequentially, in reverse order, intermittently, separately or throughconcurrent additions, to form a dispersion of the agent particles in apolymer solution.

[0084] A suitable polymer solution contains between about 1% (w/w) andabout 30% (w/w) of a suitable biocompatible polymer, wherein thebiocompatible polymer is typically dissolved in a suitable polymersolvent. Preferably, a polymer solution contains about 2% (w/w) to about20% (w/w) polymer. A polymer solution containing about 5% to about 15%(w/w) polymer is most preferred.

[0085] A suitable polymer solvent, as defined herein, is solvent inwhich the polymer is soluble but in which the aggregation-stabilizedparticles of agent are substantially insoluble and non-reactive.Examples of suitable polymer solvents include polar organic liquids,such as methylene chloride, chloroform, ethyl acetate, acetonemethylisobutylketone, n-butylacetate, isobutyl acetate, tetrahydrofuran,methyl acetate and ethyl citrate.

[0086] In yet another embodiment of the method of this invention, ametal cation component, not contained in the aggregation-stabilizedparticles of biologically active agent, is also dispersed within thepolymer solution to modulate the release of the biologically activeagent.

[0087] It is understood that a metal cation component and theaggregation-stabilized particles can be dispersed into a polymersolution sequentially, in reverse order, intermittently, separately orthrough concurrent additions.

[0088] Alternately, a polymer, a metal cation component and theaggregation-stabilized particles can be mixed into a polymer solventsequentially, in reverse order, intermittently, separately or throughconcurrent additions.

[0089] The method for forming a composition for modulating the releaseof a biologically active agent from a biodegradable polymer is furtherdescribed in co-pending U.S. patent application Ser. No. 08/237,057 andco-pending PCT Patent Application PCT/US95/05511.

[0090] One suitable method for forming a sustained release compositionfrom a polymer solution is the solvent evaporation method described inU.S. Pat. No. 3,737,337, issued to Schnoring et al., U.S. Pat. No.3,523,906, issued to Vranchen et al., U.S. Pat. No. 3,691,090, issued toKitajima et al., or U.S. Pat. No. 4,389,330, issued to Tice et al.Solvent evaporation can be used as a method to form microcarriers andother shaped sustained release devices.

[0091] In the solvent evaporation method, a polymer solution containinga dispersion of particles of an aggregation-stabilized biologicallyactive agent, is mixed in or agitated with a continuous phase, in whichthe polymer solvent is partially miscible, to form an emulsion. Thecontinuous phase is usually an aqueous solvent. Emulsifiers are oftenincluded in the continuous phase to stabilize the emulsion. The polymersolvent is then evaporated over a period of several hours or more,thereby solidifying the polymer to form a polymeric matrix having adispersion of particles of aggregation-stabilized biologically activeagent contained therein.

[0092] In this method, care must be taken not to heat the polymersolution to a temperature at which denaturing of the biologically activeagent in the aggregation-stabilized particles could occur.

[0093] Another suitable method for solidifying a polymer solution toform a polymeric matrix, containing particles of aggregation-stabilizedbiologically active agent, is the phase separation method described inU.S. Pat. No. 4,675,800, which is incorporated herein in its entirety byreference. In this method, polymer within a polymer solution containingaggregation-stabilized particles is precipitated around the particles bythe addition of a polymer non-solvent to the polymer solution to form anemulsion, wherein the polymer non-solvent is immiscible with the polymersolvent.

[0094] A preferred method for forming aggregation-stabilizedmicrocarriers from a polymer solution uses rapid freezing and solventextraction as described in U.S. Pat. No. 5,019,400, issued to Gombotz etal. and co-pending U.S. patent application Ser. No. 08/433,726, filedMay 18, 1995, the teachings of which are incorporated herein in theirentirety by reference. This method of microcarrier formation, ascompared to other methods, such as phase separation, additionallyreduces the amount of biologically active agent required to produce asustained release composition with a specific content and also minimizesthe loss of biological activity during microparticle formation. Furtherdiscussion of the high level of biological activity, typically >98%,maintained in the microparticles of the present invention, formed usingthis preferred method, is provided in Example 2. Also see Examples 2, 5and 7 for additional descriptions of microparticle formulations by thismethod.

[0095] In this method, the polymer solution, containing the dispersionof aggregation-stabilized particles, is processed to create droplets,wherein at least a significant portion of the droplets contain polymersolution and aggregation-stabilized particles. These droplets are thenfrozen by means suitable to form microparticles. Examples of means forprocessing the polymer solution dispersion to form droplets includedirecting the dispersion through an ultrasonic nozzle, pressure nozzle,Rayleigh jet, or by other known means for creating droplets from asolution.

[0096] Means suitable for freezing droplets to form microparticlesinclude directing the droplets into or near a liquified gas, such asliquid argon and liquid nitrogen to form frozen microdroplets which arethen separated from the liquid gas. The frozen microdroplets are thenexposed to a liquid non-solvent, such as ethanol, or ethanol mixed withhexane or pentane. The solvent in the frozen microdroplets is extractedas a solid and/or liquid into the non-solvent to form microcarrierscontaining aggregation-stabilized biologically active agent. Mixingethanol with other non-solvents, such as hexane or pentane, can increasethe rate of solvent extraction, above that achieved by ethanol alone,from certain polymers, such as poly(lactide-co-glycolide) polymers.

[0097] A wide range of sizes of sustained release microcarriers can bemade by varying the droplet size, for example, by changing theultrasonic nozzle diameter. If very large microcarriers are desired, themicrocarriers can be extruded through a syringe directly into the coldliquid. Increasing the viscosity of the polymer solution can alsoincrease microparticle size. For example, the size of the microcarriersproduced by this process can vary over a wide range, such as fromgreater than about 1000 to about 1 micrometers, or less, in diameter.

[0098] Yet another method of forming a sustained release composition,from a polymer solution, includes film casting, such as in a mold, toform a film or a shape. For instance, after putting the polymer solutioncontaining a dispersion of aggregation-stabilized particles into a mold,the polymer solvent is then removed by means known in the art, or thetemperature of the polymer solution is reduced, until a film or shape,with a consistent dry weight, is obtained. Film casting of a polymersolution, containing a biologically active agent, is further describedin co-pending U.S. patent application Ser. No. 08/237,057.

[0099] It is believed that the release of the biologically active agentcan occur by two different mechanisms. The agent can be released bydiffusion through aqueous filled channels generated in the polymericmatrix, such as by the dissolution of the agent or by voids created bythe removal of the polymer's solvent during the synthesis of thesustained release composition. A second mechanism is the release of theagent due to degradation of the polymer.

[0100] The rate of polymer degradation can be controlled by changingpolymer properties that influence the rate of hydration of the polymer.These properties include, for instance, the ratio of different monomers,such as lactide and glycolide, comprising a polymer; the use of theL-isomer of a monomer instead of a racemic mixture; the polymer endgroup; and the molecular weight of the polymer. These properties canaffect hydrophilicity and crystallinity, which control the rate ofhydration of the polymer. Hydrophilic excipients such as salts,carbohydrates and surfactants can also be incorporated to increasehydration and which can alter the rate of erosion of the polymer.

[0101] By altering the properties of the polymer, the contributions ofdiffusion and/or polymer degradation to the release of biologicallyactive agent can be controlled. For example, increasing the glycolidecontent of a poly(lactide-co-glycolide) polymer and decreasing themolecular weight of the polymer can enhance the hydrolysis of thepolymer and thus, provides an increased agent release from polymererosion.

[0102] In addition, the rate of polymer hydrolysis may be increased innon-neutral pH's. Therefore, an acidic or a basic excipient can be addedto the polymer solution, used to form the microcarriers, to alter thepolymer erosion rate.

[0103] The sustained release device of this invention can beadministered to a human, or other animal, by injection, implantation(e.g, subcutaneously, intramuscularly, intraperitoneally,intracranially, intravaginally and intradermally), administration tomucosal membranes (e.g., intranasally or by means of a suppository), orin situ delivery (e.g. by enema or aerosol spray) to provide the desireddosage of an agent based on the known parameters for treatment with thatagent of the various medical conditions.

[0104] The invention will now be further and specifically described bythe following examples.

EXAMPLE 1 FORMATION OF AGGREGATION-STABILIZED INTERFERON

[0105] IFN-α,2b, which was used in the present Examples, is identical toIFN-α,2 as described in Rubenstein et al., Biochem. Biophys. Acta, 695:705-716 (1982), with the exception that the lysine at position 23 ofIFN-α,2 is an arginine in IFN-α,2b. The IFN was stabilized by forming acomplex with Zn⁺² ions, wherein the complex has a lower solubility inaqueous solutions than does non-complexed IFN.

[0106] The IFN was complexed as follows. The IFN-α,2b was dissolved indifferent volumes of 10 mM sodium bicarbonate buffer (pH 7.2) to formIFN solutions with concentrations between 0.1 and 0.5 mM IFN. A 10 mMZn⁺² solution was prepared from deionized water and zinc acetatedihydrate and then was added to the IFN solutions to form Zn⁺²-IFNsolutions with a final IFN concentration of about 1.3 mg/ml and aZn⁺²:IFN molar ratio of 2:1, 4:1 or 10:1, respectively. The pH of theZn⁺²-IFN solution was then adjusted to 7.1 by adding 1% acetic acid. Acloudy suspended precipitate, comprising aggregation-stabilized IFNwherein the IFN is stabilized as a complex with Zn⁺², formed in eachsolution.

[0107] The suspension of aggregation-stabilized IFN was then micronizedusing an ultrasonic nozzle (Type V1A; Sonics and Materials, Danbury,Conn.) and sprayed into a polypropylene tub (17 cm diameter and 8 cmdeep) containing liquid nitrogen to form frozen particles. Thepolypropylene tub was then placed into a −80° C. freezer until theliquid nitrogen evaporated. The frozen particles, which containedZn⁺²-stabilized IFN, were then lyophilized to formaggregation-stabilized IFN particles.

EXAMPLE 2 PREPARATION OF PLGA MICROCARRIERS CONTAININGAGGREGATION-STABILIZED IFN

[0108] Samples of blocked PLGA (intrinsic viscosity of 0.15 dl/g)obtained from Birmingham Polymers (Birmingham, Ala.) or a hydrophilicunblocked PLGA (intrinsic viscosity of 0.17 dl/g) obtained fromBoehringer Ingelheim Chemicals, Inc. (Montvale, N.J.), were dissolved in10 ml of methylene chloride per gram of PLGA to form polymer solutions.To these polymer solutions were added about 0.033, 0.1 or 0.2 grams ofaggregation-stabilized IFN particles per gram of PLGA, formed asdescribed in Example 1 to form polymer solutions with the followingformulations: Zn:IFN Molar IFN:PLGA IFN:MgCO₃ IFN:ZnCO₃ Formula PLGARatio Mass Ratio Mass Ratio Mass Ratio 1 Blocked 2:1 0.2:1 N/A N/A 2Blocked 4:1 0.2:1 N/A N/A 3 Blocked 10:1  0.2:1 N/A N/A 4 Blocked 2:10.1:1 1:1 N/A 5 Unblocked 2:1 0.033:1  1:1 N/A 6 Blocked 2:1 0.033:1 N/A 3:1 7 Blocked 2:1 0.1:1 N/A 1:1 8 Blocked 2:1 0.1:1 N/A 8:1

[0109] When added to the polymer solution, MgCO₃ and ZnCO₃ were sievedthrough a 38 micrometer (#400) sieve. Each formulation was thensonicated using an ultrasonic probe (Virtis, Co., Gardiner, N.Y.) tofragment and suspend aggregation-stabilized IFN particles in the polymersolutions. The size of the sonicated, aggregation-stabilized IFNparticles was between about 2-15 microns. The suspension was then placedin a 10 ml gas-tight syringe.

[0110] About 400 ml of 100% ethanol per gram PLGA was added to a roundpolypropylene tub. This solution was frozen by surrounding the tub withliquid nitrogen. The frozen ethanol was then covered with 500 ml ofliquid nitrogen per gram of PLGA. The IFN suspension was then pumpedfrom the syringe by a syringe pump (Orion Sage Pump Model 355, OrionResearch Inc., Boston, Mass.), at a rate of 1.7 ml/min, into anultrasonic nozzle (Type V1A, Sonics and Materials, Danbury, Conn.) thatwas placed above the container containing the frozen ethanol coveredwith liquid nitrogen. The nozzle atomized the IFN suspension intodroplets which froze upon contact with the liquid nitrogen and formedmicrocarriers which sank to the surface of the frozen ethanol.

[0111] The container was placed into a −80° C. freezer, therebyevaporating the liquid nitrogen and allowing the ethanol to melt. As theethanol thawed, the microcarriers sank into it. The temperature waslowered to −95.1° C. and the methylene chloride was extracted from themicrocarriers. After 24 hours, an additional 400 ml of 100% ethanol pergram of PLGA, which was prechilled to −80° C., was added to thecontainer. Three days after the microcarriers were prepared, theethanol/microcarrier slurry was filtered using a 0.65 micron Durapore™membrane (Millipore, Bedford, Mass.). The filtered microcarriers werethen vacuum dried in a lyophilizer.

EXAMPLE 3 In vitro Release of IFN Encapsulated with Non-Metal CationStabilizer Compared to IFN Stabilized with Zn^(+2 WITH Zn) ⁺²

[0112] Dextran 70 (Spectrum Chemical Manufacturing Co., Gardena, Calif.)was added to a solution of IFN-α,2b in 10 mM sodium phosphate buffer ata weight ratio of 1:1 (Dextran:IFN). The solution was micronized throughan ultrasonic nozzle as described in Example 1 and the frozen particleswere then lyophilized. The IFN-Dextran particles were subsequentlymicroencapsulated in blocked PLGA as described in Example 2 to formIFN-Dextran microcarriers. Aggregation-stabilized IFN particles (2:1Zn⁺²:IFN ratio), as described in Example 1, were also microencapsulatedas described in Example 2 to form aggregation-stabilized IFNmicrocarriers.

[0113] In vitro dissolution was conducted on the two microcarrierformulations by incubating 20 mg of each type of microcarrier in bufferat 37° C. IFN release from the microcarriers was monitored by BioRadprotein assay (BioRad Inc. Richmond, Calif.).

[0114] IFN release from the IFN-Dextran microcarriers was linear for thefirst 10 days with an average release rate of 6.4%/day. The releasecontinued at a rate of 0.4%/day from day 10 to day 14 with a totalcumulative release of 66% by day 14. No further release of protein fromthe microcarriers was detected. The microcarriers were dried down at day28. The IFN-Dextran remaining was extracted from the microcarriers andthe protein was characterized by testing its solubility in water andmonomer content by sodium dodecyl sulfate (SDS) polyacrylamide gelelectrophoresis (PAGE). Only 18% of the protein remaining inside themicrocarriers was water soluble. The insoluble protein was solubilizedusing SDS and run on a gel. The insoluble material contained 19%covalent aggregates and 81% non-covalent aggregates.

[0115] In contrast the microcarriers with the IFN aggregation-stabilizedwith Zn⁺² showed linear release for at least 28 days at a rate of2.7%/day. The analyses indicate the formulation of IFN with zinc is morestable resulting in a longer period of continuous release of proteinfrom the microcarriers.

EXAMPLE 4 FORMATION OF AGGREGATION-STABILIZED hGH

[0116] Purified recombinant human growth hormone (hGH), whose DNAsequence is as described in U.S. Pat. No. 4,898,830, issued to Goeddelet al., was used in this Example. The human growth hormone wasstabilized by forming a complex with Zn⁺² ions, wherein the complex hasa lower solubility in aqueous solutions than does non-complexed hGH.

[0117] The hGH was dissolved in samples of a 4 mM sodium bicarbonatebuffer (pH 7.2) to form hGH solutions with concentrations between 0.1and 0.5 mM hGH. A 0.9 mM Zn⁺² solution was prepared from deionized waterand zinc acetate dihydrate and then was added to the hGH solutions toform Zn⁺²-hGH solution. The pH of the Zn⁺²-hGH solution was thenadjusted to between 7.0 and 7.4 by adding 1% acetic acid. A cloudysuspended precipitate, comprising Zn⁺²-stabilized hGH formed.Lyophilized, aggregation-stabilized hGH particles were then formed asdescribed in Example 1.

EXAMPLE 5 PREPARATION AND ANALYSIS OF PLGA MICROCARRIERS CONTAININGBIOLOGICALLY ACTIVE, AGGREGATION-STABILIZED hGH

[0118] Microcarriers containing aggregation-stabilized hGH, formed asdescribed in Example 4, were prepared using the method of Example 2 fromhydrophilic unblocked PLGA (50:50 PLGA, 9,300 Daltons; RG502H polymer;Boehringer Ingelheim Chemicals, Inc.), blocked PLGA (50:50 PLGA, 10,000Daltons; Lot #115-56-1, Birmingham Polymers, Inc., Birmingham, Ala.) andunblocked PLGA (50:50 PLGA, 31,000 Daltons; RG503H, Boehringer IngelheimChemicals, Inc.) and varying amounts of ZnCO₃.

[0119] The integrity of the hGH encapsulated in microcarriers wasdetermined by extracting the hGH from the microcarriers. Themicrocarriers were placed in a tube containing methylene chloride andvortexed at room temperature to dissolve the polymer. Acetone was thenadded to the tube, which was subsequently vortexed, to extract andcollect the hGH. The collected hGH was then freeze-dried andre-constituted in HEPES buffer containing 10 mM EDTA. Appropriatecontrols were run to ensure that the extraction process did not affectthe integrity of the protein.

[0120] The integrity of the encapsulated hGH was analyzed by measuringthe percent of hGH monomer contained in the hGH sample afterencapsulation by size exclusion chromatography (SEC).

[0121] The results of SEC analyses of the hGH integrity of hGH sustainedrelease microcarriers were provided below. Formulation (polymer; % ZincCarbonate) % Monomer (SEC) 31K unblocked; 6% ZnCO3 98.6 31K unblocked;6% ZnCO3 99.2 31K unblocked; 3% ZnCO3 97.7 31K unblocked; 3% ZnCO3 97.831K unblocked; 1% ZnCO3 97.6 31K unblocked; 0% ZnCO3 97.8 31K unblocked;0% ZnCO3 97.1 10K blocked; 1% ZnCO3 98.2 10K blocked; 1% ZnCO3 98.4 8Kunblocked; 0% ZnCO3 98.5 10K blocked; 1% ZnCO3 98.4

[0122] The results showed that the encapsulation process did not causeaggregation of the protein.

EXAMPLE 6 FORMATION OF AGGREGATION-STABILIZED EPO

[0123] Erythropoietin was derived as described in U.S. Pat. No.4,703,008. The EPO was dissolved in deionized water to form an aqueoussolution having a concentration of approximately 1 mg/ml. Differentsamples of the EPO solution were then dialyzed against three changes ofthe appropriate formulation buffer (i.e., 5 mM phosphate buffer (pH 7),5 mM citrate buffer (pH 7), 5 mM citrate/5 mM phosphate buffer (pH 7) or10 mM bicarbonate buffer (pH 7)).

[0124] Following dialysis, the concentration of EPO in the dialyzedsolutions was verified to be approximately 1 mg/ml as determined bymeasuring absorbance at 280 nm (ε=1.345 L gm⁻¹ cm⁻¹).

[0125] Portions of the dialyzed EPO solutions were then mixed withconcentrated solutions of candidate anti-aggregation agents (i.e.,ammonium sulfate, zinc acetate, mannitol/sucrose or mannitol/maltose) toform the EPO formulations provided in Table I below. The candidateanti-aggregation agent solutions also possibly contained additionalexcipients (i.e, inulin, glycine and TWEEN 20™ surfactant).

[0126] The anti-aggregation agent solutions were separately prepared inthe same buffers used to dialyze the EPO solutions to which they weresubsequently added.

[0127] Approximate volumes of each anti-aggregation agent solution andof additional buffer were added to a 50 ml polypropylene tube to achievethe desired concentrations for the formulations (described in Table I).Each dialyzed EPO solution was then added to the appropriateanti-aggregation agent solution and then the solutions were mixed bygentle inversion. TABLE I Formulations (wt %) Am1 Am4 Am7 Ma1 Ma3 Ma4Zn1 Zn6 EPO 10.0 10.1 9.9 10.0 10.0 10.0 10.0 10.0 Ammonium 66.8 64.779.1 0.0 0.0 0.0 0.0 0.0 Sulfate Zinc Acetate 0.0 0.0 0.0 0.0 0.0 0.076.9 76.9 Mannitol 0.0 0.0 0.0 62.5 52.5 72.5 0.0 0.0 Sucrose 0.0 0.00.0 10.0 0.0 10.0 0.0 0.0 Maltose 0.0 0.0 0.0 0.0 10.0 0.0 0.0 0.0 5 mMCitrate 0.0 15.1 0.0 0.0 0.0 0.0 0.0 0.0 Buffer (pH 7) 5 mM 0.0 0.0 10.07.5 7.5 7.5 0. 0.0 Phosphate Buffer (pH 7) 5 mM Citrate/ 22.1 0.0 0.00.0 0.0 0.0 0.0 0.0 5 mM Phosphate Buffer (pH 7) 10 mM 0.0 0.0 0.0 0.00.0 0.0 13.1 12.1 Bicarbonate Buffer (pH 7) Inulin 1.1 10. 1.0 0.0 0.00.0 0.0 0.0 Glycine 0.0 0.0 0.0 10.0 10.0 0.0 0.0 0.0 TWEEN 20 ™Surfactant 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0

[0128] Lyophilized, aggregation-stabilized EPO particles were thenformed from the EPO solutions as described in Example 1. The EPOparticles were removed from the lyophilizer under an atmosphere of drynitrogen, handled in a low humidity environment, and stored desiccatedat −80° C.

EXAMPLE 7 PREPARATION AND ANALYSIS OF PLGA MICROCARRIERS CONTAININGAGGREGATION-STABILIZED ERYTHROPOIETIN

[0129] Microcarriers containing the aggregation-stabilized EPOformulations of Example 6 were prepared from unblocked (50:50; MW 10,000Daltons) PLGA, obtained from Boehringer Ingelheim Chemicals, Inc.,Montvale, N.J., or blocked (50:50; MW 10,000 Daltons) PLGA obtained fromBirmingham Polymers, Inc., Birmingham, Ala.

[0130] In addition, microcarriers, containing the Am7 formulation ofaggregation-stabilized EPO particles, were prepared from unblocked(50:50) PLGA with a molecular weight of approximately 31,000 Daltons or45,000 Daltons, (Boehringer Ingelheim Chemicals, Inc., Montvale, N.J.).

[0131] The method described in Gombotz et al. (U.S. Pat. No. 5,019,400),and in Example 2, was used to encapsulate the aggregation-stabilized EPOparticles of Example 6 in PLGA. In each case, polymer was dissolved in5.1 ml of methylene chloride to form a polymer solution. Magnesiumcarbonate, or zinc carbonate, was sieved through a 38 micrometer sieveand was then added to the polymer solution to a final concentration of10% w/vol. The polymer/salt suspension was subsequently combined with 30mg of aggregation-stabilized EPO particles.

[0132] The polymer solution, containing suspended salt and EPOparticles, was placed in an ice-water bath and sonicated using anultrasonic probe (Virtis Co., Gardiner, N.Y.) to reduce the proteinparticle size to approximately 2 to 3 micrometers in diameter and toform a dispersion of EPO particles within the polymer solution.

[0133] Microcarriers containing aggregation-stabilized EPO were preparedusing the method described in Example 2.

[0134] The immunoreactivity of the EPO in these sustained releasemicrocarriers was subsequently determined by extracting protein andanalyzing by radioimmunoassay (RIA) (Incstar: Stillwater, Minn.). Toextract the EPO from the microcarriers, approximately 10 mg ofmicrocarriers were placed in a tube with 250 μl of methylene chloride.The samples were vortexed for 10 to 20 seconds and left at roomtemperature for 5 minutes to dissolve the polymer. A sample of acetone(750 μl) was added, vortexed for an additional 10 seconds, andcentrifuged at 14,000 rpm for 30 seconds at 4° C. to pellet the EPO. Thesupernatant was removed and the methylene chloride and acetone stepswere repeated twice more. Samples were dried in a lyophilizer or vacuumoven for 14-18 hours at room temperature. The EPO pellet wasreconstituted in 1 ml HEPES buffer by vortexing for about 10 seconds,then standing at room temperature for about 1 hour until completelydissolved. The extracted EPO was diluted in buffer (8.1 mM Na₂HPO₄, 1.5mM KH₂PO₄, 400 mM NaCl, pH 7.5) to a concentration of approximately 25μg/ml for analysis.

[0135] The immunoreactivity of the EPO was found to be 121,000±5000units per mg of EPO. This specific activity is comparable to the rangeobtained for bulk EPO (130,000-140,000 units per mg of EPO) thus showingan insignificant reduction of EPO activity due to the method of formingthe sustained release compositions of the present invention. Monomercontent was found to be greater than 98% for all microcarriers.

[0136] The microcarriers containing Am1 and Am7 EPO particles were alsoassayed for EPO dimer, by size exclusion chromatography (SEC), and forhigh molecular weight EPO aggregates by SDS-PAGE/Western blot analysis.No EPO dimer or high molecular weight aggregates were detected.

EXAMPLE 8 In Vitro Release of EPO from Aggregation-Stabilized EPOMicrocarriers

[0137] The in vitro release kinetics of EPO from aggregation-stabilizedparticles within PLGA microcarriers were assessed in HEPES buffer (75 mMHEPES, 115 mM NaCl, 0.1% (by volume) TWEEN 20™, 0.1% (by weight) sodiumazide titrated to pH 7.4 with NaOH) or in HEPES buffer containing 2% or20% sheep serum. The studies were conducted by suspending 8-10 mg ofmicrocarriers in 1-5 ml of buffer at 37° C. At specified time points,the buffer was removed in full and replaced with fresh buffer.

[0138] In samples incubated in HEPES buffer, the releases over time ofEPO monomer (biologically active EPO) and of EPO aggregates(biologically inactive EPO) were determined by size exclusionchromatography (SEC). The results of the SEC analyses upon in vitrorelease kinetics in HEPES buffer of various microcarriers, wherein themicrocarriers were a) unblocked PLGA (MW 10,000 Daltons) microcarrierscontaining formulations Am1 or Am7, and b) blocked PLGA (MW 10,000Daltons) microcarriers containing Zn1, are provided in FIGS. 1, 2 and 3,respectively. FIGS. 1 and 2 show the EPO released from formulationscontaining ammonium sulfate as an anti-aggregation agent was almost allmonomeric EPO over the length of the release period.

[0139]FIG. 3 shows the EPO released from a formulation containing zincacetate, as an anti-aggregation agent, contained significant levels ofaggregate which increased substantially over the length of the releaseperiod.

[0140] The results of the SEC and RIA analyses upon in vitro releasekinetics in HEPES buffer, and in HEPES/serum, of various microcarriers(all in 10,000 Dalton PLGA) which contained different EPO formulationsof Example 6 are provided in Table II. The initial burst and releaserate were determined in the HEPES/serum test by RIA. The integrity ofthe released EPO was assessed in HEPES buffer by SEC. TABLE II AggregateEPO Released Initial Average Release Load (% init. Burst ReleaseDuration Formula (%) Polymer/Salt Load) (%) (%/day) (days) Zn1 10Blocked/ 12 66 1.2 14 10% MgCO₃ Zn1 10 Blocked/ 22 46 1.7 28 10% ZnCO₃Zn6 10 Blocked/ 37 32 1.6 28 10% ZnCO₃ Am1 5 Unblocked/ 1 39 1.4 21 10%MgCO₃ Am1 10 Blocked/ 2 71 0.3 3 10% MgCO₃ Am4 5 Unblocked/ 1 29 1.1 2110% MgCO₃ Am4 5 Unblocked/ 1 35 0.9 28 none Ma1 5 Unblocked/ 1 44 1.8 2410% MgCO₃ Ma3 10 Unblocked/ 1 71 1.3 21 10% MgCO₃ Ma4 10 Blocked/ 1 770.6 3 10% ZnCO₃

[0141] These analyses show that the addition of suitableanti-aggregation agents significantly reduced the aggregation of EPOover the release periods. These analyses also demonstrated that theaddition of a metal cation component (e.g., salt) to the polymer, aswell as the selection of the type of polymer (i.e., blocked orunblocked) significantly affected the initial burst level and theduration of release.

EXAMPLE 9 Integrity of EPO Released In Vitro from Aggregation-StabilizedEPO Microcarriers

[0142] The purpose of the experiment was to determine the integrity ofEPO released from PLGA microcarriers having varying concentrations ofammonium sulfate.

[0143] Aggregation-stabilized EPO formulations comparable to Am7, excepthaving 10%, 20%, or 40% ammonium sulfate, were prepared as described inExample 6. The eliminated ammonium sulfate was replaced with sodiumchloride or sucrose such that the total weight of ammonium sulfate andsodium chloride or sucrose was 79%.

[0144] The percent monomeric and aggregate EPO were determined after 35days and 42 days release in vitro. The Am7 formulation, as well as the40% ammonium sulfate/NaCl formulation produced 3-4% aggregates at bothtime points, whereas the 10% and 20% ammonium sulfate/NaCl formulationsproduced 5-6% aggregates. Mannitol formulations produced results similarto the 10% and 20% ammonium sulfate formulations.

[0145] In the case where ammonium sulfate was replaced with sucrose,there was not sufficient drug released from the 40% ammonium sulfateformulation to quantitate. The 10% and 20% ammonium sulfate formulationswith sucrose, like their sodium chloride counterparts, showed moreaggregates (6-9%) than were observed with the Am7 formulation.

EXAMPLE 10 In Vivo Release of Aggregation-Stabilized IFN-α,2b fromPolymeric Microcarriers in Rats

[0146] Microcarriers, containing aggregation-stabilized IFN, which wereprepared as described in Example 2, were tested in rats for the in vivorelease of IFN-α,2b. Normal rats were obtained from Taconics, Inc.(Germantown, N.Y.). The animals were fed with a standard diet andallowed free access to water. Three to four rats were injectedsubcutaneously in the interscapular region with a dose of 0.6-2.0 mg ofIFN/kg, in a 0.5% gelatin, 1% glycerol and 0.9% w/w NaCl vehicle, on day0 for each of the IFN microcarriers of Example 2. Blood samples weretaken from the tail vein of each rat at 1, 2, 4, 8, 10 (optionally), 24,36 and 48 hours after injection. Additional blood samples were thentaken approximately once a day for the following 4-5 days. The IFNconcentration in the rat serum samples was determined using an IFN-αimmunoradiometric assay, (Celltech, Slough, U.K), hereinafter “IRMA”.The IRMA assay has a minimum limit of detecting of 6 IU/ml. The IFNserum levels for control rats, which did not receive the microcarrierscontaining Zn⁺²-stabilized IFN were found to be less than 6 IU/ml.

[0147] The results of the IRMA assays conducted on the rats receivingthe microcarriers of Example 2 are shown in FIGS. 4-10. FIGS. 4-10 showthat these injectable microcarrier formulations provided a sustainedrelease of immunologically active IFN-α.

EXAMPLE 11 In Vivo Release of Aggregation-Stabilized IFN from PolymericMicrocarriers in Immunosuppressed Rats

[0148] One group of male Sprague-Dawley rats (N=2) (control group),weighing 400±50 g (S.D.) was injected as described in Example 10 withthe microcarriers of Formula 8 of Example 2. An additional group (N=2)of rats (test group) was also given daily intraperitoneal injections of10 mg cyclosporin A (Sandimmune® Injection, Sandoz, East Hanover, N.J.)and 5 mg hydrocortisone (Spectrum Co., Gardena, Calif.) in 0.5 mlsterilized saline for injection (USP) per Kg of body weight for days 0to 14 and then injections twice a week for days 15 to 28. Theseinjections were to suppress the response of the rats' immune systems tothe release of IFN-α,2b in vivo. No antibody titers were detected inthese rats for the duration of treatment.

[0149] This method of immunosuppression is further described inco-pending U.S. patent application Ser. No. 08/480,813, filed Jun. 7,1995.

[0150] The control group did not receive injections to suppress theirimmune response to IFN-α,2b. Antibodies were detected after day 7 inthese rats.

[0151] The serum levels of IFN-α,2b in the rats of the experimentalgroup and the control group were determined by IRMA through day 29 (696hours and 480 hours, respectively). These results are provided in FIG.11. The results for both groups are the same through day 7 suggestingthat the cyclosporin A/hydrocortisone treatment does not affect themeasured serum concentrations of IFN. The results show that the controlgroup serum levels measured for IFN were artificially high due to theirproduction of antibodies to the IFN-α,2b. The results for theexperimental group, in which antibody formation was suppressed, showedsustained release of IFN-α,2b for up to at least 29 days for thepreferred microcarriers (Formula 8) of Example 2.

EXAMPLE 12 In Vivo Release of IFN-α,2b from Aggregation-Stabilized IFNMicrocarrier in Monkeys

[0152] Microcarriers (Formula 8), as prepared as in Example 2, weretested in a test group of four male cynomolgous monkeys (Charles RiverPrimates) for release of IFN-α,2b. The animals were fed with a standarddiet and allowed free access to water. Each monkey was injectedsubcutaneously with a dose of about 0.12 mg IFN/kg monkey on day zero.

[0153] Concurrently, each monkey in a control group of four monkeys,with the same diet and water access as the test group, weresubcutaneously injected with an aqueous saline solution containing about0.12 mg IFN/kg monkey.

[0154] Blood samples were taken from the femoral vein at 0, 1, 3, 6, 12,24, 48, 96, 120, 144, 168, 240, and 336 hours after injection. TheIFN-α,2b concentration in the monkey serum samples was determined usingboth a cytopathic effect assay (CPE; Pharmacopeial Previews, UnitedStates Convention, Inc., November-December 1990, page 1241) and IRMA.The CPE results for both groups are provided in FIG. 12.

[0155] For the test group, the IRMA and CPE results were similar andshowed sustained release of IFN-α,2b from the microcarriers.

[0156] The CPE and IRMA results for the control group, which receivedthe aqueous IFN-α,2b injection, showed that the IFN-α,2b concentrationfell below detectable limits before the second day of testing.

[0157]FIG. 12 shows that the microcarrier formulation injected providedsustained release of biologically active IFN-α.

EXAMPLE 13 Assay for hGH after in Vivo Degradation ofAggregation-Stabilized HGH Microcarriers

[0158] Microcarriers of blocked-PLGA, containing 15% w/w Zn⁺²-stabilizedhGH and 0%, 6%, 10% or 20% ZnCO₃ were formed by the method of Example 5.Groups of test rats were injected subcutaneously with 50 mg samples ofthe different hGH microcarriers. The rats were sacrificed after 60 daysand the skin samples were excised from the injection sites.

[0159] The excised skin samples were placed in 10% Neutral BufferedFormalin for at least 24 hours. They were then trimmed with a razorblade to remove excess skin and placed in PBS.

[0160] Tissue samples were processed by Pathology Associates, Inc.(Frederick, Md.). The skin samples were embedded in glycomethacrylate,sectioned and assayed for the presence of hGH using aHistoScan/LymphoScan Staining Kit (Product #24-408M; Accurate Chemical &Scientific Corp., Westbury, N.Y.) according to the manufacturer'sinstructions. Tissue samples were scored for the presence or absence ofstaining which was indicative of the presence or absence of hGH in thesample.

[0161] All skin samples, associated with hGH microcarrier injections,tested positive for the presence of hGH thus indicating that theblocked-PLGA microcarriers still contained hGH after 60 days in vivo.

[0162] The method described in Example 5 was used to form microcarriersby encapsulating 0% or 15% w/w hGH, in the form of Zn:hGH complex, andalso 0%, 1% or 6% w/w ZnCO₃ salt, within blocked-PLGA and withinunblocked-PLGA.

[0163] In vivo degradation of unblocked-PLGA microcarriers versusblocked-PLGA microcarriers were compared by injecting samples ofmicrocarriers into rats and then analyzing the microcarriers remainingat the injection site at various times post-injection. Three rats wereassayed at each time point for each microcarrier sample. On the day ofadministration of the microcarriers, 750 μl of vehicle (3% carboxymethylcellulose (low viscosity) and 1% Tween-20 in saline) was added to vialscontaining 50±1 mg of microcarriers. Immediately, the vials were shakenvigorously to form a suspension which was then aspirated into a 1.0 ccsyringe without a needle.

[0164] Rats (Sprague-Dawley males) were anesthetized with a halothaneand oxygen mixture. The injection sites (intrascapular region) wereshaven and marked with a permanent tatoo to provide for the preciseexcision of skin at the sampling time points. Each rat was injected withan entire vial of microcarriers using 18 to 21 gauge needles.

[0165] On designated days (days 15, 30, 59 and 90 post-injection foranimals receiving blocked-PLGA microcarriers, or days 7, 14, 21, 28 and45 post-injection for animals receiving unblocked-PLGA microcarriers)the rats were sacrificed by asphyxiation with CO₂ gas and the skin atthe injection sites (including microcarriers) was excised. Since themicrocarriers tended to clump at the injection sites, the presence orabsence of microcarriers was determined visually.

[0166] The visual inspections found that the unblocked-PLGAmicrocarriers degraded substantially faster than the blocked-PLGAmicrocarriers, and that the addition of ZnCO₃ to the blocked-PLGAsubstantially slowed polymeric degradation. For example, in the ratsinjected with unblocked-PLGA microcarriers containing 0% hGH and 0% or1% ZnCO₃, no microcarriers were visible on day 21. In addition, for ratsinjected with blocked-PLGA microcarriers containing 0% hGH and 0% ZnCO₃,a few microcarriers were visible on day 60 and none were visible on day90. Furthermore, for rats injected with blocked-PLGA microcarrierscontaining 0% or 15% hGH and 6% ZnCO₃, microcarriers were visible on day90.

EXAMPLE 14 In Vivo Release of Aggregation-Stabilized Hgh Microcarriersin Rats

[0167] Studies were conducted in rats to screen various hGH microcarrierformulations, determine pharmacokinetic parameters following intravenous(IV), subcutaneous (SC) and SC osmotic pump (ALZET®) administration ofhGH, and to evaluate serum profiles and in vivo release rate of varioushGH microcarrier formulations.

[0168] Sprague-Dawley rats were divided into groups of three each,randomized by body weight, and one hGH microcarrier formulation wasadministered to each group. Rats were injected subcutaneously withapproximately 7.5 mg of hGH in 50 mg of microcarriers, suspended in 0.75ml of an aqueous injection vehicle. The vehicle composition was 3% CMC(low viscosity), 1% Polysorbate 20, in 0.9% NaCl. The microcarrier dosedelivered was determined indirectly by weighing the residual dose in theinjection vial and correcting for residual injection vehicle. The hGHdose was then computed from the protein loading of the microcarriersdetermined by nitrogen analysis.

[0169] Blood samples were collected at predetermined intervals for up to10 days after injection. Blood samples of 250 μl were collected duringthe first 24 hours and at least 400 μl at time points after 24 hours.Blood samples were clotted and hGH concentrations in serum weredetermined using a radio-immuno assay (RIA) using an RIA kit from ICN.

[0170] For the determination of pharmacokinetic parameters, hGH insaline was administered to rats by subcutaneous bolus injection,intravenously or delivered via an osmotic pump which was implantedsubcutaneously.

[0171] Three groups of rats received single subcutaneous injections ofhGH in 0.9% NaCl at 0.5 or 7.5 mg/kg at a dose volume of 1.0 ml/kg andtwo groups received single intravenous bolus injections of hGH in 0.9%NaCl solution at about 1.0 mg and 5.0 mg of hGH per kg rat with a dosevolume of 1.0 ml/kg. For the ALZET® pump study, rats were divided intofour groups of three rats each, randomized by body weight and dosed withabout 20 mg/ml and 40 mg/ml hGH in 0.9% saline solution loaded intopumps (ALZET® Model 2002, 200 μl, 14 days release), and with about 4mg/ml and 12 mg/ml hGH in 0.9% saline solution loaded into pumps (ALZET®Model 2ML4, 2 ml, 28 days release). Expected release rates from thepumps correspond to about 2% and 4 to 6% of the ProLease hGH dose (about15 mg/kg) per day, respectively. The ALZET® pumps were implantedsubcutaneously in the inter-scapular region after soaking for 1-2minutes in sterile saline.

[0172] The formulations of hGH sustained release microcarriers,synthesized as described in Example 5 contained 15% w/w hGH complexedwith Zn in a ratio of 6:1 Zn:hGH; 0%, 1%, 3% or 6% w/w zinc carbonate;and 8K unblocked PLGA, 10K blocked PLGA or 31K unblocked PLGA.

[0173] To evaluate the various hGH sustained release formulations, Cmax,Cd5 and Cmax/Cd5 were the in vivo indices used, where Cmax is themaximum serum concentration observed, and Cd5 is the serum concentrationat day 5 which should approximate the steady state concentration. Theresults were as follows: ‘Burst’ in % Monomer Cmax C day 5 Cmax/CssFormulation vitro (%) Day 7 (ng/ml) (ng/ml) 8K PLGA 22.0 ± 0.9 99.3*323.3 ± 98.6 20.4 ± 14.2 19.5 ± 10.6 unblocked 0% ZnCO3 8K PLGA 16.4 ±1.6 97.3* 309.0 ± 67.1 20.4 ± 14.2 39.5 ± 17.7 unblocked 1% ZnCO3 8KPLGA 15.9 ± 6.9 98.7  670.5 ± 244.4 9.0 ± 4.2 44.8 ± 22.6 unblocked 3%ZnCO3 8K PLGA 17.6 ± 2.7 99.3 358.0 ± 58.9 18.8 ± 14.7 42.4 ± 6.8 unblocked 6% ZnCO3 31K PLGA 12.3 ± 1.1 98.2   592 ± 318.2 4.5 ± 1.5132.5 ± 47.9  unblocked 0% ZnCO3 31K PLGA 11.4 ± 1.3 98.8 432.7 ± 91.65.1 ± 0.3 84.1 ± 14.9 unblocked 1% ZnCO3 31K PLGA  7.9 ± 1.9 99.4  643.6± 203.9 8.0 ± 2.6 93.3 ± 62.0 unblocked 3% ZnCO3 31K PLGA 15.8 ± 0.599.8 1691.8 ± 340.0 6.6 ± 0.8 262.2 ± 83.5  unblocked 6% ZnCO3 10K PLGA12.7 ± 0.1 99.3  615.9 ± 384.3 4.5 ± 1.0 155.0 ± 126.8 unblocked 1%ZnCO3 10K PLGA 18.1 ± 3.2 99.6 1053.2 ± 293.3 3.6 ± 0.8 291.7 ± 71.1 blocked 3% ZnCO3 10K PLGA  9.9 ± 1.4 99.0 1743.5 ± 428.4 4.9 ± 2.7 561.1± 361.6 blocked 6% ZnCO3

[0174] The results of the screening showed that the two unblocked (8Kand 31K) polymers had different in vivo release kinetics compared to theoriginal formulation, which used blocked 10K PLGA and 6% w/w zinccarbonate. Cmax values were generally lower with the unblocked polymerformulations than with the original formulation which suggested that thein vivo ‘burst’ may be lower with the unblocked polymer formulations.The ‘burst’ was defined as the percent of hGH released in the first 24hours after injection. The in vitro ‘burst’ values were between 8-22%.The zinc carbonate content of the formulations did not appear to have aneffect on the ‘burst’ or the in vitro release profile.

[0175] The serum concentrations between days 4 and 6 were maintained ata fairly constant level above baseline (or the pre-bleed levels) withthe unblocked polymer formulations, while serum concentrations with theblocked formulations, at the same time points were close to the baselinelevels. The in vitro release data for up to 7 days showed that thereleased hGH protein was monomeric. Useful data could not be obtainedbeyond day 6 because of anti-hGH antibody formation in the rats.

EXAMPLE 15 In Vivo Release of hGH from Aggregation-Stabilized hGHMicrocarriers in Immunosuppressed Rats

[0176] Two groups of male Sprague-Dawley rats (N=3) (control groups),weighing 400±50 g (S.D.) were injected as described in Example 14 withthe microcarriers of Example 5. Two additional groups (N=3) of rats(test groups) were also given daily intraperitoneal injections of 10 mgcyclosporin A and 5 mg hydrocortisone in 0.5 ml sterilized saline forinjection (USP) per kg of body weight for days 0 to 14 and theninjections three times a week for days 15-28. No antibody titers weredetected in these rats for the duration of treatment.

[0177] The control group did not receive injections to suppress theirimmune response to hGH. Antibodies were detected after day 6 in theserats.

[0178] The serum levels of hGH in the rats of the experimental groupsand the control groups were determined by RIA through day 28. Theseresults are provided in FIGS. 13 and 14. The results for both pairs ofcontrol and experimental groups were the same through day 6 suggestingthat the cyclosporin A/hydrocortisone treatment did not affect themeasured serum concentrations of hGH. The results further show that thecontrol groups' serum levels of hGH were artificially high due to theirproduction of antibodies to hGH.

[0179] The results for the experimental groups, in which antibodyformation was suppressed, showed sustained release of hGH for up to 24days and 26 days for the 31K unblocked PLGA and 8K blocked PLGAmicrocarriers, respectively, of Example 5.

EXAMPLE 16 In Vivo Release of hGH from Aggregation-Stabilized hGHMicrocarriers in Rhesus Monkeys

[0180] The objective of this primate study was to evaluate thepharmacokinetic profiles of different hGH sustained release formulationsas compared to more traditional methods of administering hGH (e.g.,bolus sc injections, daily sc injections and sc injection combined withthe use of an osmotic pump) and to determine which hGH sustained releaseformulation gave the optimal hGH blood concentration profile.

[0181] The formulations for the hGH sustained release microcarrierstested were 1) 15% hGH (complexed with Zn⁺² at a 6:1 Zn⁺²:hGH ratio), 6%w/w zinc carbonate and 10K blocked PLGA; 2) 15% hGH (complexed with Zn⁺²at a 6:1 Zn⁺²:hGH ratio), 1% w/w zinc carbonate and 8K unblocked PLGA(“RG502H” PLGA polymer); and 3) 15% hGH (complexed with Zn⁺² at a 6:1Zn⁺²:hGH ratio), 1% w/w zinc carbonate and 31K unblocked PLGA (“RG503H”PLGA polymer). The microcarriers were formed as described in Example 5.

[0182] There were four monkeys per group and each animal received asingle subcutaneous injection into the dorsal cervical region on Day 1.A dose of 160 mg of hGH sustained release microcarriers (24 mg of hGH)was administered to each monkey in 1.2 ml of injection vehicle through a20 gauge needle. The injection vehicle was an aqueous vehicle containing3% w/v low viscosity Carboxymethyl Cellulose (sodium salt), 1% v/v Tween20 (Polysorbate 20) and 0.9% sodium chloride.

[0183] The hGH dose was intended to provide measurable hGH serumconcentrations for pharmacokinetic analysis. To obtain pharmacokineticparameters, additional study groups of four monkeys each were included,specifically 1) a single subcutaneous injection (24 mg hGH), 2) dailysubcutaneous injections (24 mg/28 days=0.86 mg hGH/day), 3) asubcutaneous injection (3.6 mg hGH) combined with an ALZET®osmotic pump(20.4 mg hGH)(total dose of 24 mg hGH), and 4) a subcutaneous injectionof the injection vehicle as a control (only used 3 monkeys for thevehicle control group).

[0184] The osmotic pump gave sustained serum hGH levels comparable tothe hGH microcarriers up to day 28 as programmed to release hGH. Thepumps were removed on day 31.

[0185] Blood samples were collected at the following times for hGH andIGF-1 analyses: −7, −5, −3 days, pre-dose and, 0.5, 1, 2, 3, 5, 8, 10,12, 24, 28, 32 and 48 hours, 5, 4, 6, 8, 11, 14, 17, 20, 23, 26, 29, 32,25, 28, 41, 44, 47, 50, 53, 56 days post-dose.

[0186] The concentrations of IGF-1, which is expressed when a body hasan effective serum level of hGH, and hGH in the serum were thenmeasured. An IRMA kit from RADIM (distributed by: Wein Laboratories,P.O. Box 227, Succasunna, N.J.) was used to quantify hGH in monkeyserum. The IRMA assay had a limit of quantification in PBS buffer of 0.1ng/mL and in pooled juvenile rhesus monkey serum of 1.5 ng/mL with abasal GH level of about 4 ng/mL. RIA was used to quantify the IGF-1serum levels.

[0187] The results of the hGH serum level assays for the 10K blockedPLGA, 8K unblocked PLGA and 31K unblocked hGH microcarriers of Example 5are provided in FIGS. 15-17, respectively. Further, the results of thehGH and IGF-1 serum assays for the 8K unblocked PLGA microcarriers ofExample 5 are shown in FIG. 18.

[0188] In addition, a comparison of the results of the IGF-1 serumassays for the 8K unblocked PLGA microcarriers of Example 5 as comparedto the serum levels for daily subcutaneous injections of hGH are shownin FIG. 19.

[0189] The results showed that the hGH sustained release microcarrierswere releasing significant, sustained levels of hGH over a one monthperiod while the subcutaneous injections were not able to maintain thesame serum levels.

[0190] The IGF-1 serum profile showed that serum IGF-1 concentrationswere elevated above the baseline values between days 2 and 29 afteradministering the microparticles. This shows that enough hGH was beingreleased from the hGH sustained release microcarriers to cause apharmacodynamic effect. This also indicates that the hGH released wasbiologically active which suggests that the encapsulation process hadnot adversely affected the biopotency of hGH.

EXAMPLE 17 In Vivo Release of Aggregation-Stabilized EPO from PolymericMicrocarriers in Immunosuppressed Rats

[0191] Male Sprague-Dawley rats, weighing 400±50 g (S.D.), were used asthe animal model. The rats were not fasted before the experiments andsubsequently were fed with a standard diet, an iron supplement, andallowed free access to water. Iron dextran (Sigma Co., St. Louis, Mo.) 5mg/kg was injected intraperitoneally twice a week.

[0192] These experiments utilized the immunosuppression method describedin Examples 11 and 15 for suppressing antibody production in the testanimals in response to the EPO released (or injected) to obtain accurateprofiles of serum EPO levels.

[0193] The purpose of the first experiment was to compare the in vivopharmacodynamic effects of aggregation-stabilized EPO released fromsustained release microcarriers to EPO injected subcutaneously as abolus, specifically upon serum reticulocyte profiles. Two groups ofthree rats were injected subcutaneously in the interscapular region onday 0 with 10,000 units of RMAm7 EPO microcarriers (unblocked 10K PLGAcontaining 10% MgCO₃ and 5% Am7) and subsequently on day 28 with a 2,000unit bolus of aqueous EPO. The control group did not receive thecyclosporin A/hydrocortisone therapy, which the test group did receive.

[0194] Blood samples were taken from the tail vein of each rat at 1, 3,4, 8, 10, 14, 16, 20, 24, 28, 30 or 31, 32 and 36 hours after injection.Additional blood samples were then taken approximately twice a week forthe following 5 weeks.

[0195] Blood reticulocyte levels were counted for selected blood sample.The results are provided in FIG. 20. FIG. 20 shows higher reticulocytecounts in immunosuppressed rats in response to both theaggregation-stabilized EPO microcarriers and the EPO bolus. Thenon-immunosuppressed rats (control group) showed lower reticulocytelevels due to antibody formation resulting from the immune systems'responses to EPO. This is particularly shown by the lack of asignificant increase in reticulocyte levels in the control group afterreceiving the EPO bolus on day 28.

[0196]FIG. 20 also shows that injection with sustained releasemicrocarriers resulted in a longer period of elevated serum reticulocytelevels than did a bolus of EPO.

[0197] The purpose of the second experiment was to compare the in vivopharmacokinetic and pharmacodynamic effects of EPO released from varioussustained release microcarriers.

[0198] The rats in each of four groups rats (N=3) were injectedsubcutaneously in the interscapular region with one of four of thefollowing formulations of microcarriers: RMAm1 Unblocked 10K PLGA/10%MgCO₃/5% Am1 RMMa1 Unblocked 10K PLGA 10% MgCO₃/5% Ma1 PZZn1 Blocked 10KPLGA/10% ZnCO₃/5% Zn1 RMAm7 Unblocked 10K PLGA/10% MgCO₃/5% Am7

[0199] Each rat received between 10,000 to 12,000 units per animal. Eachrat was also given daily an intraperitoneal injection of 10 mg ofcyclosporin A and 5 mg of hydrocortisone.

[0200] Blood samples were taken from the tail vein of each rat at 1, 2,4, 8, 10 (optionally), 24, 36 and 48 hours after injection. Additionalblood samples were then taken approximately once a day for the following10 days and approximately two times per week for the next two weeks. TheEPO concentration in the rat serum samples was determined using byELISA. In addition, blood reticulocyte levels were counted.

[0201] Serum EPO and blood reticulocyte profiles for these formulationsare provided in FIGS. 21 and 22. EPO levels remained above baseline inthese animals for approximately 14 days, showing a sustained release ofbiologically active EPO. Elevated reticulocyte levels were observed forabout 17 days. Further, the response of immature and total reticulocytelevels were proportional and not significantly different from each otherfollowing EPO treatment.

EXAMPLE 18 EFFECT OF ZINC CARBONATE ON RELEASE LEVELS OFAGGREGATION-STABILIZED IFN-α,2b IN RATS

[0202] Rats (N=4) in three test groups were injected, as described inExample 9, with the microcarriers of formulas 4 and 6-8 of Example 2.The dose of IFN for each rat was about 0.8 mg/kg.

[0203] The purpose of the test was to determine if the initial burst andsustained level of IFN-α,2b released in vivo can be varied by changingthe weight ratio of zinc carbonate to IFN-α,2b in microcarriers.

[0204] The weight ratio of zinc carbonate to IFN in microcarriers testedfor initial burst effects were 0:1, 1:1, 3:1 and 8:1. Blood samples werethen taken from the tail vein of each rat at 1, 2, 4, 8, 12, 24, 32, 48,72, 96, 120, 144 and 168 hours after injection. The IFN-α,2bconcentrations in the rat serum samples were determined by IRMA. Thetests found that the addition of zinc carbonate to the formulationreduces initial burst in vivo. Specifically, initial bursts measured, asa percentage of the total IFN in the microcarriers which were releasedover the first 24 hours, for microcarriers having weight ratios of 0:1,1:1, 3:1 and 8:1 were 35±13%, 23±7%, 13±5% and 8±1%, respectively.

[0205] These initial burst results suggest that the amount of metalcation in the polymer can be used to vary the burst.

[0206] For the sustained release test, the weight ratio of zinccarbonate to IFN in microcarriers tested were 1:1, 3:1 and 8:1. Thesustained release results of this test are presented in FIG. 23. Thesustained level observed for Formula 7 of Example 1, having a weightratio of 1:1, was 250±30 IU/ml during days 5-7. The level observed forFormula 6, having a weight ratio of 3:1, was 180±10 IU/ml during days5-7, whereas that for a Formula 8, having a weight ratio of 8:1, was110±10 IU/ml.

[0207] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A device for the sustained release in vivo of awater soluble, biologically active agent wherein said agent issusceptible to aggregation, comprising: a) a drug delivery device; andb) aggregation-stabilized, biologically active agent wherein saidaggregation-stabilized agent is disposed within the drug deliverydevice.
 2. A sustained release device of claim 1 wherein the drugdelivery device is a biocompatible polymeric matrix.
 3. A sustainedrelease device of claim 1 wherein the aggregation-stabilized,biologically active agent includes a biologically active agent and anaggregation-stabilizer.
 4. A sustained release device of claim 3 whereinthe aggregation-stabilizer is at least one salting-out salt.
 5. Asustained release device of claim 3 wherein the aggregation-stabilizeris a metal cation component.
 6. A sustained release device of claim 5wherein the aggregation-stabilizer and a metal cation of the metalcation component are complexed.
 7. A sustained release device of claim 3wherein the aggregation-stabilizer is a buffer.
 8. A sustained releasedevice of claim 3 wherein the aggregation-stabilizer is polyethyleneglycol.
 9. A sustained release device of claim 3 wherein theaggregation-stabilized, biologically active agent is in particulateform.
 10. A composition for the sustained release in vivo of a watersoluble, biologically active agent wherein said agent is susceptible toaggregation, comprising: a) a biocompatible polymer; and b)aggregation-stabilized, biologically active agent wherein saidaggregation-stabilized agent is disposed within the polymer.
 11. Asustained release composition of claim 10 wherein theaggregation-stabilized, biologically active agent includes abiologically active agent and an aggregation-stabilizer.
 12. A sustainedrelease composition of claim 10 wherein the biologically active agentand the aggregation-stabilizer are mixed.
 13. A sustained releasecomposition of claim 10 wherein the aggregation-stabilizer reduces thesolubility of the protein in an aqueous fluid.
 14. A composition ofclaim 13 wherein the anti-aggregation agent is a salting-out salt.
 15. Acomposition of claim 14 wherein the salting-out salt comprises a saltcontaining a cation selected from the group consisting Mg⁺², Li⁺, Na⁺,K⁺, NH₄ ⁺ and combinations thereof.
 16. A composition of claim 13wherein the salting-out salt comprises a salt containing an anionselected from the group consisting of SO₄ ⁻², HPO₄ ⁻², acetate, citrate,tartrate, CL⁻, NO₃ ⁻, CLO₃ ⁻, I⁻, CLO₄ ⁻, SCN⁻ and combinations thereof.17. A composition of claim 13 wherein the salting-out salt is ammoniumsulfate.
 18. A composition of claim 11 wherein the aggregationstabilizer is mannitol.
 19. A sustained release composition of claim 11wherein the aggregation-stabilizer is a buffer.
 20. A sustained releasecomposition of claim 11 wherein the aggregation-stabilizer is a metalcation from a metal cation component.
 21. A sustained releasecomposition of claim 11 wherein the metal cation of said metal cationcomponent is a biocompatible multivalent cation.
 22. A sustained releasecomposition of claim 12 wherein the multivalent metal cation is selectedfrom the group consisting of Zn⁺², Ca⁺², Cu⁺², Mg⁺² and combinationsthereof.
 23. A sustained release composition of claim 10 wherein thebiocompatible polymeric matrix is formed of a biodegradable polymer. 24.A sustained release composition of claim 20 further comprising a secondmetal cation component, wherein the second metal cation component isdispersed within the biocompatible polymeric matrix.
 25. A sustainedrelease composition of claim 24 wherein the second metal cationcomponent is selected from the group consisting of magnesium hydroxide,magnesium carbonate, calcium carbonate, zinc carbonate, magnesiumacetate, zinc acetate, magnesium sulfate, zinc sulfate, magnesiumchloride, zinc chloride, zinc citrate, magnesium citrate and acombination thereof.
 26. A composition for the sustained release in vivoof a water soluble, biologically active agent aggregation, comprising:a) a biodegradable polymer; and b) aggregation-stabilized, biologicallyactive agent wherein said aggregation-stabilized agent is disposedwithin the biodegradable polymer.
 27. A sustained release composition ofclaim 26 wherein the biodegradable polymer ispoly(lactide-co-glycolide).